Maize DWF4 gene and expression vector, application and plant thereof

A technology for expressing vectors and genes, applied in the fields of molecular biology and biology

Inactive Publication Date: 2011-09-14
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the DWF4 gene has been studied relatively deeply in other species, there is no repo...

Method used

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  • Maize DWF4 gene and expression vector, application and plant thereof
  • Maize DWF4 gene and expression vector, application and plant thereof
  • Maize DWF4 gene and expression vector, application and plant thereof

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Experimental program
Comparison scheme
Effect test

Embodiment approach ( 1

[0022] Embodiment (1): Cloning method of maize DWF4 gene

[0023] (1). Extraction of RNA: TRIZOL kit was used to extract total RNA of young leaves of maize inbred line Qi 319;

[0024] (2). Synthesis of the first strand of cDNA (using Transgen company cDNA first strand synthesis kit method)

[0025] Add the following reagents to a 0.25ml centrifuge tube:

[0026] mRNA

5μl

Oligo d(T)

1μl

Buffer

10μl

EasyScript Reverse Transcriptase

1μl

RNAase free water

3μl

[0027] After mixing by pipetting, take a water bath at 42°C for 30 minutes, then a water bath at 85°C for 10 minutes, cool on ice for 5 minutes, centrifuge lightly, and store for later use.

[0028] (3). 5' tailing reaction of cDNA

[0029] 1. Reaction system:

[0030] cDNA

20μl

5×TdT Buffer

10μl

0.1%BSA

5μl

100mM dCTP

1μl

TdT

1μl

wxya 2 O Up to

50μl

[0031] 2.37°C for 30 minute...

Embodiment approach ( 3

[0047] Embodiment (three): the construction of overexpression vector, refer to image 3 A;

[0048] (1) Construction of PZP211: UBI vector: The pTCK303 vector containing the Ubqutin promoter and the pZP211 empty vector were respectively digested with restriction enzymes Hind III and BamH I, and the target fragment was detected by electrophoresis and recovered. The cleaved Ubqutin promoter and the pZP211 empty vector fragment were ligated with T4 ligase, and the operation steps were carried out according to the instructions of T4DNA ligase produced by Fermentas Company. Then the ligation product was transformed into Escherichia coli DH5α competent cells, and cultured overnight on LB solid medium containing spectinomycin (50mg / L). White colonies were picked and cultured overnight in LB liquid medium containing spectinomycin (50mg / L). Plasmid DNA was extracted by alkaline method and identified by enzyme digestion.

[0049] (2) According to the obtained corn gene sequence, desig...

Embodiment approach ( 4

[0057] Embodiment (four): Agrobacterium-mediated transformation of immature maize embryos and acquisition of resistant plants

[0058] (1) Pick Agrobacterium (a single colony of Agrobacterium carrying a recombinant plasmid) one day before dipping and inoculate it in YEP medium containing 50mg / L spectinomycin, shake the bacteria overnight at 28°C and 250rpm, precipitate the next morning and use AB Induction medium resuspension, at least 5 hours of incubation before precipitation, infiltration medium resuspension and infiltration.

[0059] (2) The immature corn embryos 10-13 days after pollination were taken and placed in the pre-dipping medium.

[0060] (3) Centrifuge at 2700 rpm for 5 seconds, and discard the medium.

[0061] (4) Add 2ml of pre-dipping medium to resuspend, and centrifuge at 2700rpm for 5 seconds.

[0062] (5) 46°C water bath for 3 minutes, ice bath for 1 minute, and centrifuge at 2700 rpm for 5 seconds. Discard the medium and add 2ml of pre-dipping medium. ...

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Abstract

The invention discloses a maize DWF4 gene and an expression vector, application and a plant thereof. In a method for breeding a high-yield plant provided by the invention, DWF4 genes are introduced into a receptor plant for over-expression to obtain a transgenic crop with superior agronomic characters. Measurement of various indexes of the transgenic plant discovers that the transgenic plant grows vigorously, is obviously heightened and has enhanced photosynthesis, delayed aging of blades and increased grain weight compared with a non-transgenic plant. The improvement of the agronomic characters can be used for increasing yield and quality of the crop and has important economical value and social benefit.

Description

technical field [0001] The invention relates to a key gene for regulating the biosynthesis of brassinosteroid-maize DWF4 gene and its expression vector, application and plant, belonging to the field of molecular biology and biotechnology. Background technique [0002] Maize is not only the third largest food crop in the world, but also an important monocot model plant for basic and applied research. With the completion of the sequencing of the maize genome, the research on maize genetics, genomics and molecular biology has increasingly become a current hotspot. The genetic transformation technology of maize has become one of the core means in the field of maize research. The research on transgenic maize began in the early 1980s. From the first report of transgenic maize to the present for more than 20 years, the research on maize transformation receptors, transformation methods and selection of marker genes has developed rapidly. At present, a variety of excellent corn var...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/63A01H5/00
Inventor 张宪省赵翔宇申强
Owner SHANDONG AGRICULTURAL UNIVERSITY
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